HOXB7 IN LUNG CANCER: A NOVEL ROLE IN STEM CELL AND IPS BIOLOGY

Current diagnostic tools do not allow prognostic evaluation of patients with early stage lung cancer or selection of patients that might benefit from adjuvant chemotherapy. Therefore, the identification of novel prognostic markers in early-stage lung cancer is paramount. In this scenario, the transcription factor HOXB7, belonging to the homeobox family, has been shown to correlate with poor prognosis in different types of cancer and recently also in stage I lung adenocarcinoma. To better understand the prognostic implication of alterations in HOXB7 expression in lung cancer, we performed a bioinformatics analysis of multiple lung cancer expression datasets in order to identify gene sets representing cancer-relevant biological functions enriched in high-HOXB7 expressing tumors. We found several gene sets enriched in high-HOXB7 expressing tumors representing molecular mechanisms involved in epithelial to mesenchymal transition, in cancer progression, and, interestingly, in stemness and cellular reprogramming. Based on these results, we hypothesized that HOXB7 may have a role in the expansion of the stem cell compartment in cancer, a mechanism that has been shown to be a hallmark of enhanced tumorigenicity and of increased metastatic potential. Analysis of the stem-related surface marker CD90 revealed that overexpression of HOXB7 in lung cells increases the CD90high sub population. CD90high, but not CD90low cells, are able to form spheroids, which is an hallmark of stemness. Indeed, the sphere forming efficiency of normal lung BEAS-2B cells was 22% and 1.64% in CD90high and CD90low populations, respectively. In addition, we found that silencing of LIN28B counteracts the expansion of the CD90high population. LIN28B was recently described as an oncogene that regulates the cancer stem cell compartment. We found that LIN28B is under the direct transcriptional control of HOXB7. Therefore, we propose a novel molecular mechanism driven by HOXB7 and can increase stem-like properties in lung cells. We further demonstrated that the HOXB7-LIN28B axis plays an important role in reprogramming of adult cells into induced pluripotent stem cells (iPS). Indeed, HOXB7 may enhance the reprogramming efficiency achieved by the three genes OCT4, KLF4, SOX2 in both mouse embryonic fibroblast and human epithelial BEAS-2B cells by substituting MYC in the transcription factor cocktail of reprogramming factors used by Yamanaka. Of note, LIN28B silencing strongly decreases the number of reprogrammed colonies in high-HOXB7 expressing cells. These findings suggest that HOXB7, through transcriptional induction of the LIN28B gene, activates a program relevant for stem/iPS cell biology and for tumor progression, possibly opening a new line of research for the development of more effective therapies for metastatic lung cancer patients

The Potential of Omega-3 Supplementation to Reduce Muscle-Inflammation after Muscle-Damaging Exercise

Muscle Damaging exercise (EIMD) induces inflammation and relates to strength loss, muscle-soreness and impaired recovery. Overall, this means a performance impairment which might be relevant for those who engages in competitions or strenuous physical activities. It remains unclear whether Omega-3 fatty acids (O-3) supplementation blunts the exercise-induced inflammation associated with EIMD and therefore prevents performance impairment. PURPOSE: Following a three-week supplementation with O-3, indirect markers of muscle damage were examined after a bout of EIMD to determine if supplementation had any beneficial effect in maintaining leg-strength levels. METHODS: Eight healthy, recreationally active caucasian males (28.13 ± 3.4 yr) were randomly allocated to a supplementation group (SUP, n = 4) to receive 2.85g/day O-3 supplementation or a control group (CON, n = 4) for three weeks. Following supplementation, participants performed a bout of EIMD, which consisted of performing 10 sets of 15 repetitions of leg extension at a self-assessed intensity of 7/10 on the Rate of Perceived Exertion scale. Creatine Kinase (CK) from venous blood samples, isometric right-leg strength, squat-jump test and perceived soreness were determined, as indirect markers of muscle-damage at Baseline, immediately after EIMD (POST) and 48 hours after EIMD to coincide with the delayed muscle inflammatory response. RESULTS: No statistically significant differences were found between Baseline and POST. There was a trend for smaller increase of CK levels (pre vs 48-h post EIMD) on the SUP group (38.8% increase) compared with the CON group (105.6% increase; P = 0.051). There was no significant effect (baseline vs. 48-h post EIMD) on muscle strength between SUP and CON group (P > 0.05), however, CON showed a larger decrease in strength compared to SUP (> 6.3% vs SUP). No differences in jump height were found between SUP and CON (P > 0.05). There was no significant difference in muscle soreness at 48-h post EIMD between SUP and CON group (P = 0.171). CONCLUSION: Three weeks of O-3 supplementation might decrease exercise-induced muscle inflammation after eccentric exercise. The lack of statistical significance may be adduced to the limitations of the study design and sample size. Supplementation with O-3 can be beneficial in athletes undergoing heavy exercise regimes and in sedentary individuals restarting physical activity, decreasing the exercise related muscle inflammation. The encouraging results from this pilot study have led to designing further work related to this topic

Acidic environments trigger intracellular H+-sensing FAK proteins to re-balance sarcolemmal acid-base transporters and auto-regulate cardiomyocyte pH

AIMS: In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely-balanced sarcolemmal acid-base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge and return pHi to normal. METHODS AND RESULTS: Following left-ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl–/HCO3- exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinisation when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate to improve systemic buffering had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes (NRVMs) incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) upregulation and Slc4a2 (AE2) downregulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3- and became ablated when acidic incubation media had low chloride concentration, a manoeuvre that reduces the extent of pHi decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterised as pH-sensors, ablated pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. CONCLUSIONS: Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities. TRANSLATIONAL PERSPECTIVE: As a consequence of the inherent thermodynamic coupling between intra- and extracellular pH (pHi/pHe), sustained changes to perfusion, such as those in coronary disease or development, would have deleterious effects on the internal acid-base milieu of myocytes and hence cardiac function, unless offset by a corrective process. Using in-vivo and in-vitro models of acidification, we characterise this adaptive process functionally, and describe how it is engaged to auto-regulate pHi. This additional layer of homeostatic oversight enables the myocardium to operate within its optimal pHi-range, even at times when vascular perfusion is failing to maintain chemical constancy of the interstitial fluid

The dopamine D1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium

Background Dopamine receptors comprise two subgroups, Gs protein-coupled “D1-like” receptors (D1, D5) and Gi-coupled “D2-like” receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression. Methods We evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively. Results The dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells. Conclusions These results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms

HPV16 E7-Dependent Transformation Activates NHE1 through a PKA-RhoA-Iinduced Inhibition of p38alpha

Background: Neoplastic transformation originates from a large number of different genetic alterations. Despite this genetic variability, a common phenotype to transformed cells is cellular alkalinization. We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 (HPV16), that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na/H-+(+) exchanger isoform 1 (NHE1) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice.Methodology: Here, we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1.Principal Findings: We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell. Further, using pharmacological agents and transient transfections with dominant interfering, constitutively active, phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1, we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein, RhoA, and its subsequent inhibition.Conclusions: All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation

Components of the mitochondrial cAMP signalosome

3′-5′-Cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signalling is activated by different extracellular stimuli and mediates many diverse processes within the same cell. It is now well established that in order to translate into the appropriate cellular function multiple extracellular inputs, which may act simultaneously on the same cell, the cAMP/PKA signalling pathway is compartmentalised. Multimolecular complexes are organised at specific subcellular sites to generate spatially confined signalosomes, which include effectors, modulators and targets of the pathway. In recent years, it has become evident that mitochondria represent sites of compartmentalised cAMP signalling. However, the exact location and the molecular composition of distinct mitochondria signalosomes and their function remain largely unknown. In this review, we focus on individual components of the cAMP/PKA signalling pathway at distinct mitochondria subdomains represented by the outer and inner mitochondrial membranes, the intermembrane space and the matrix, highlighting some of the questions that remain unanswered

A barter economy in tumors: exchanging metabolites through gap junctions

To produce physiological functions, many tissues require their cells to be connected by gap junctions. Such diffusive coupling is important in establishing a cytoplasmic syncytium through which cells can exchange signals, substrates and metabolites. Often the benefits of connectivity become apparent solely at the multicellular level, leading to the notion that cells work for a common good rather than exclusively in their self-interest. In some tumors, gap junctional connectivity between cancer cells is reduced or absent, but there are notable cases where it persists or re-emerges in late-stage disease. Diffusive coupling will blur certain phenotypic differences between cells, which may seem to go against the establishment of population heterogeneity, a central pillar of cancer that stems from genetic instability. Here, building on our previous measurements of gap junctional coupling between cancer cells, we use a computational model to simulate the role of connexin-assembled channels in exchanging lactate and bicarbonate ions down their diffusion gradients. Based on the results of these simulations, we propose that an overriding benefit of gap junctional connectivity may relate to lactate/bicarbonate exchange, which would support an elevated metabolic rate in hypoxic tumors. In this example of barter, hypoxic cancer cells provide normoxic neighbors with lactate for mitochondrial oxidation; in exchange, bicarbonate ions, which are more plentiful in normoxic cells, are supplied to hypoxic neighbors to neutralize the H+ ions co-produced glycolytically. Both cells benefit, and so does the tumor

Local modulation of Cystic Fibrosis Conductance Regulator: cytoskeleton and compartmentalised cAMP signalling.

The cystic fibrosis conductance regulator (CFTR) is a cAMP-regulated Cl(-) channel expressed predominantly at the apical membrane of secreting epithelial cells. Mutations in the CFTR gene lead to cystic fibrosis, the most frequent genetic disease in the Caucasian population. The most common mutation, a deletion of phenylalanine at position 508 (F508del), impairs CFTR folding and chloride channel function. Although an intense effort is underway to identify compounds that target the F508del CFTR structural defect and promote its expression and stability at the plasma membrane, so far their clinical efficacy has proven to be poor, highlighting the necessity to better understand the molecular mechanism of CFTR regulation and of the pathogenesis of the disease. Accumulating evidence suggests that the inclusion of the CFTR in macromolecular complexes and its interaction with the cortical cytoskeleton may play a key role in fine-tuning the regulation of channel function. Here we review some recent findings that support a critical role for protein-protein interactions involving CFTR and for the cytoskeleton in promoting local control of channel activity. These findings indicate that compounds that rescue and stabilise CFTR at the apical membrane may not be sufficient to restore its function unless the appropriate intracellular milieu is also reconstituted

Local modulation of cystic fibrosis conductance regulator: cytoskeleton and compartmentalized cAMP signalling.

The cystic fibrosis conductance regulator (CFTR) is a cAMP-regulated Cl(-) channel expressed predominantly at the apical membrane of secreting epithelial cells. Mutations in the CFTR gene lead to cystic fibrosis, the most frequent genetic disease in the Caucasian population. The most common mutation, a deletion of phenylalanine at position 508 (F508del), impairs CFTR folding and chloride channel function. Although an intense effort is under way to identify compounds that target the F508del CFTR structural defect and promote its expression and stability at the plasma membrane, so far their clinical efficacy has proven to be poor, highlighting the necessity to better understand the molecular mechanism of CFTR regulation and of the pathogenesis of the disease. Accumulating evidence suggests that the inclusion of the CFTR in macromolecular complexes and its interaction with the cortical cytoskeleton may play a key role in fine-tuning the regulation of channel function. Here we review some recent findings that support a critical role for protein-protein interactions involving CFTR and for the cytoskeleton in promoting local control of channel activity. These findings indicate that compounds that rescue and stabilize CFTR at the apical membrane may not be sufficient to restore its function unless the appropriate intracellular milieu is also reconstituted